Technical Field of the Invention
[0001] The invention relates to anti-freeze polypeptides (AFPs) and food product containing
AFPs.
Background to the Invention
[0002] Anti-freeze polypeptides (AFPs) have been suggested for improving the freezing tolerance
of foodstuffs.
[0003] For the purpose of the invention, the term AFP has the meaning as well-known in the
art, namely those proteins which inhibit the growth of ice-crystals. See for example
US 5,118,792.
[0004] WO 90/13571 discloses antifreeze peptides produced chemically or by recombinant DNA
techniques from plants. The AFPs can suitably be used in food-products such as ice-cream.
Example 3B shows modified ice-crystal shapes if a water-ice mixture is frozen into
a film in combination with 0.01 wt% of AFP.
[0005] WO 92/22581 discloses AFPs from plants which can be used for controlling ice crystal
shape in ice-cream. This document also describes a process for extracting a polypeptide
composition from intercellular spaces of plants by infiltrating leaves with an extraction
medium without rupturing the plant cells.
[0006] WO 94/03617 discloses the production of AFPs from yeast and their possible use in
ice-cream. WO 96/11586 describes fish AFPs produced by microbes.
[0007] Up till now, however the use of AFPs has not been applied to commercially available
consumer products. One reason for this are the high costs and complicated process
for obtaining AFPs. Another problem is that sources of the AFPs are either difficult
to obtain in sufficient quantities (e.g. fish containing AFPs) or are not directly
suitable for use in food products.
[0008] The present invention aims to provide novel antifreeze polypeptides which have the
advantage that they can easily be obtained from an abundant natural source and which
provide good properties to products in which they are used.
[0009] It has been found that antifreeze polypeptides which possess good recrystallisation
inhibition properties can be obtained from carrots. In particular it has been found
that antifreeze polypeptides obtained from carrots show markedly better properties
as compared to polypeptides isolated from other root vegetables. In particular the
antifreeze polypeptides of the invention are capable of providing good recrystallisation
inhibition properties without significantly changing the crystal shape of the ice-crystals,
therewith possible leading to more favourable properties e.g. soft ice-cream.
[0010] Applicants have found that the effective antifreeze polypeptides from carrots are
generally characterised by an apparent Molecular Weight on SDS-PAGE of 36 kDa.
[0011] In this context it will be clear to the skilled person that due to variation e.g.
in SDS PAGE, the apparent molecular weight can only be determined with some variation
in the results. For the purpose of the invention these variations e.g. from 30 to
40 kDa or from 34 to 38 kDa are also embraced within the scope of the term "apparent
Molecular Weight of 36 kDa".
[0012] Applicants also have found that the effective anti-freeze polypeptides comprise fragments
having an amino acid sequence as represented in the
examples.
[0013] The complete amino acid sequence of the preferred AFP of the invention is represented
below. Accordingly, the invention relates to an anti-freeze protein having an amino
acid sequence as shown in Listing 1:
[0014] Also embraced within the invention are isoforms and derivatives of the above mentioned
polypeptides which still possess the antifreeze properties and which show at least
75% homology with the polypeptide of Listing 1 more preferred more than 85%, most
preferred more than 95%. For the purpose of the invention the term derivative also
embraces modified polypeptides which still possess the antifreeze properties, for
example glycosylated forms of the above polypeptides.
[0015] Also embraced within the invention are nucleotide sequences encoding the amino acids
as described above. In particular the invention relates to nucleotide sequences of
Listing 1 and alleles thereof.
[0016] Also embraced within the invention are nucleotide fragments derived from the coding
region that are capable of hybridizing to related genes that code for anti-freeze
peptides.
[0017] Although the proteins of the invention can easily directly be isolated from carrots,
also genetic manipulation techniques may be used to produce the proteins described
in the invention.
[0018] An appropriate host cell or non-human organism would be transformed by a gene construct
that encodes the desired polypeptide. The nucleotide sequence coding for the polypeptide
can be inserted into a suitable expression vector containing the necessary elements
for transcription and translation and in a manner that they will be expressed under
appropriate conditions (eg in proper orientation and correct reading frame and with
appropriate targeting and expression sequences). The methods required to construct
these expression vectors are well known to those skilled in the art.
[0019] A number of expression systems may be utilised to express the polypeptide coding
sequence. These include, but are not limited to, bacteria, yeast, insect cell systems,
plant cell culture systems and plants all transformed with the appropriate expression
vectors. Yeast, plants and plant culture systems are preferred in this context.
[0020] A wide variety of plants and plant cell systems can be transformed with the nucleic
acid constructs of the polypeptides. Preferred embodiments would include, but are
not limited to, maize, tomato, tobacco, carrots, strawberries, rape seed and sugar
beet.
[0021] One preferred embodiment of the invention relates to the use of AFPs of the invention
for increasing the frost tolerance of plants. This case for example be done by the
above method whereby the plants are transformed to ensure (increased) production of
the AFPs of the invention, therewith increasing the frost tolerance of said plants.
[0022] The invention also relates to antibodies which specifically bind an (epitope of the)
polypeptides of the invention.
[0023] Based on the above information it is also possible to genetically modify other natural
sources such that they produce the advantageous AFPs as identified here-above.
[0024] Preferably those AFPs are chosen which have significant ice-recrystallisation inhibition
properties. A suitable test for determining the recrystallisation inhibition properties
is indicated in example I. Also preferably AFPs in accordance to the invention provide
a ice particle size in the frozen product(mean crystal length) upon recrystallisation
of less than 50 µM, more preferred from 5 to 40 µm.
[0025] The AFPs can conveniently be used in several products, preferably in food products
which are frozen or intended to be frozen. Carrots which comprise the AFP at naturally
occuring levels are not embraced within the scope of the invention. However, food
product containing (parts) of carrots are embraced within this term. Also embraced
are carrots which have been transformed to over express the AFP of the invention i.e.
which contain the AFP at significantly higher levels than non-transformed carrots.
[0026] Examples of such food products are: frozen food products such as vegetables, sauces,
soups, snacks, frozen confectionery products such as ice-cream or water-ice, dairy
products etc.
[0027] The preferred products wherein the AFPs are used are or frozen vegetables or frozen
confectionery products such as ice-cream or water-ice. Preferably the level of AFPs
is from 0.00001 to 0.5 wt% based on the final product.
[0028] If dry-mixes or concentrates are used, the concentration may be higher in order to
ensure that the level in the final frozen product is within the above ranges. Surprisingly
it has been found that compositions of the invention can contain very low amounts
of AFPs while still being of good quality.
[0029] Preferred levels of AFP are from 0.00001 to 0.5 wt%, more preferred 0.00005 to 0.3
wt%, most preferred 0.0001 to 0.2 wt%.
[0030] For the purpose of the invention it is not necessary to add the AFP in purified form
to the food product. Also possible is to add a composition comprising AFPs e.g. an
extract of the natural material which produces the AFP.
Also it is possible to modify the food product such that the AFP is produced in situ
e.g. by adding genetically modified micro-organisms which are capable of producing
the AFP in the food product, or even to genetically modify the food product (e.g.
the vegetable) such that (the vegetable) in itself it is capable of producing the
AFP in situ.
[0031] For the purpose of the invention the term frozen confectionery product includes milk
containing frozen confections such as ice-cream, frozen yoghurt, sherbet, sorbet,
ice milk and frozen custard, water-ices, granites and frozen fruit purees.
[0032] Preferably a the level of solids in the frozen confection (e.g. sugar, fat, flavouring
etc) is more than 3 wt%, more preferred from 10 to 70wt, for example 40 to 70 wt%.
[0033] Frozen confectionery products according to the invention can be produced by any method
suitable for the production of frozen confectionery. Especially preferably however
all the ingredients of the formulation are fully mixed before the freezing process
starts.
EXAMPLES
Example I
[0034] Carrots (Daucus carota cv Autumn King) were grown in individual pots. When plants
were approximately twelve weeks old, they were transferred to a cold room and held
at 4°C in constant light during 4 weeks for cold-acclimation. Plants ... were watered
three times a week.
[0035] Fresh tissue of the carrots were ground with a pestle and mortar (cooled to 4°C)
in an equal volume buffer A (10mM EDTA, 20 mM Ascorbic acid, buffered with Tris to
pH 7.4) held on ice. The homogenates were filtered through one layer of muslin and
kept on ice prior to further use.
[0036] As a comparison several other root-plants were grown and homogenates prepared from
the roots as above.
[0037] Anti-freeze activity was measured using a modified "splat assay" (Knight et al, 1988).
2.5 µl of the solution under investigation in 30% (w/w) sucrose was transferred onto
a clean, appropriately labelled, 16 mm circular coverslip. A second coverslip was
placed on top of the drop of solution and the sandwich pressed together between finger
and thumb. The sandwich was dropped into a bath of hexane held at -80°C in a box of
dry ice. When all sandwiches had been prepared, sandwiches were transferred from the
-80°C hexane bath to the viewing chamber containing hexane held at -6°C using forceps
pre-cooled in the dry ice. Upon transfer to -6°C, sandwiches could be seen to change
from a transparent to an opaque appearance. Images were recorded by video camera and
grabbed into an image analysis system (LUCIA, Nikon) using a 20x objective. Images
of each splat were recorded at time = 0 and again after 30-60 minutes. The size of
the ice-crystals in both assays was compared. If the size at 30-60 minutes is similar
or only moderately increased (say less than 20% increased, more preferred less than
10% increased, most preferred less than 5 % increased) compared to the size at t=0,
this is an indication of good ice-crystal recrystallisation inhibition properties.
[0038] Results: from the sandwich splat assay test it appeared that samples from carrot
roots, carrot stem and carrot leaves possess significant ice-recrystallisation inhibition
properties, whereby the roots and leaves are most active. As a comparison a sample
of non-acclimated carrot roots was tested, which showed significant less activity.
For the following examples root tissue was used for further testing on carrots.
[0039] As a comparison several other vegetable roots were investigated by means of the sandwich
splat assay test in 30% sucrose. Among these vegetables were turnip, kale, brussels
sprout, wintergreen cabbage, rape, pak choi, parsnip and strawberry. None of these
sources of material provided significant ice-recrystallisation inhibition activity.
Example II
[0040] Carrot root tissue was homogenized in three volumes (w/v) buffer (20mM ascorbic acid,
10 mM EDTA, 50 mM Tris/HCL, pH 7.2) in a pre-cooled pestle and mortar and filtered
through one layer of muslin. The filtrate was centrifuged at 6,000 g, ten minutes
at 4°C; the supernatant was collected and centrifuged at 100,000g for 1 hour at 4°C.
The 100,000 g supernatant from this step is termed the soluble fraction and the pellet
the microsomal fraction.
[0041] The supernatant was applied to a 30 ml fast flow Q Sepharose (Pharmacia) column pre-equilibrated
in 50 mM Tris/HCL pH 7.4 at a flow rate of 5 ml/min supplied by a HiLoad pump P-50
controlled by a Gradifrac low pressure chromatography system (Pharmacia) at 4°C and
the eluate monitored at OD 280 by a UV monitor (Monitor UV1, Pharmacia) recorded on
a chart recorder (REC 102, Pharmacia). 5 ml fractions were collected. The column was
washed with 50mM Tris/HCL pH 7.4 at the same flow rate until the OD 280 returned to
zero. A 150ml gradient of 0-0.4 M NaCl in Tris/HCL pH 7.4 was then applied followed
by a 2 M NaCl column wash. Eluate fractions were subjected to the splat assay as in
example I.
[0042] Fractions containing anti-freeze activity as evidenced by recrystallisation inhibition
were pooled and concentrated using polyethylene glycol as follows: the fractions were
transferred in 10kDa cut off dialysis tubing (Sigma) which had been washed in tap
water, boiled in 50mM EDTA pH 7.5 for 10 minutes and rinsed in milli Q water. The
dialysis tubing containing the sample to be concentrated was covered with solid polyethylene
glycol compound Mol. Wt. 15,000 - 20,000 (Sigma) and incubated at 4°C for up to 4
hours or until the sample volume inside the dialysis tubing had reduced up to 10 fold.
[0043] The pooled concentrate from the Q sepharose column was applied either to a phenyl
Sepharose column, a SMART superdex 75 gel permeation column or an FPLC superdex 75
gel permeation column.
[0044] Carrot root anti-freeze proteins were purified by gel permeation chromatography as
follows:
[0045] 20µl aliquots of sample were applied to a SMART superdex 75 column (Pharmacia) pre-equilibrated
in 50mM Tris/HCl pH7.4 containing 0.15M NaCl (Buffer E) at a flow rate of 40µl/min
and components separated by gel permeation at the same flow rate in equilibration
buffer. The eluate was monitored at OD 280 and OD 215. 80µl fractions were collected
between 0.85 and 0.89ml, 40µl fractions between 0.89 and 1.24ml and 100µl fractions
between 1.24 and 3.0 ml. The void volume (Vo) of the column was 0.91 ml as determined
by the retention volume of a solution of Blue Dextran. The superdex column was calibrated
by application of 10µl of a solution containing 5mg/ml BSA (Mr 66kDa, retention (Ve)=1.02
ml), 3mg/ml Carbonic anhydrase (Mr 29 kDa, Ve=1.22 ml), 2mg/ml Cytochrome C (Mr 12.4
kDa, Ve=1.41 ml) and 2mg/ml Aprotinin (Mr 6.5 kDa, Ve=1.59 ml) and a standard curve
plotted of Ve/Vo against log Mr. Fractions containing anti-freeze activity were identified
by the splat assays as described in Example I, with an activity peak that showed a
retention volume of 1.16 ml and an apparent molecular weight of 40 kDa. These measurement
confirmed that the 36 kDa band from cold acclimatised carrots was an anti-freeze peptide.
[0046] SDS-PAGE was carried out according to Laemmli (1970) using the Biorad mini system.
Samples to be analyzed by SDS-PAGE were dissolved in SDS-PAGE sample buffer (Laemmli
1970), heated for 5 minutes at 100°C on a dry heating block (Techne) and centrifuged
for 3 minutes at 10,000g at room temperature. Samples (10-50µl) were applied to mini-gels
(Biorad, 0.75,1.0 or 1.5mm thickness, 10,12,15% acrylamide or 10-20% gradient acrylamide
(pre-poured from Biorad)) and electrophoretically separated. Separated polypeptides
were fixed and stained in the gel either with Coomassie blue (0.1% {w/v} Coomassie
Brilliant Blue in acetic acid/methanol/miliQ water {5:4:31, by vol}) or silver stained
using the Biorad silver stain kit according to the manufacturer's instructions. Gels
were dried between two sheets of Gelair cellophane in a Biorad gelair dryer according
to the manufacturer's instructions. Sigma high and low range molecular weight marker
kits were used according to the manufacturer's instructions for determination of apparent
M
r on SDS-PAGE.
[0047] The ion exchange chromatography was carried out with cold acclimatised carrot root
and non-cold acclimatised carrot root. The resulting gel SDS-PAGE gels showed the
presence of a 36kDa band in the cold acclimatised sample. This band was much less
abundant in the non-cold acclimatised root. This 36kDa band was hence attributed to
anti-freeze activity.
Example III
[0048] For protein sequencing, the 36kDa carrot root protein was purified as described in
the previous example and then to ensure further purification the sample to be sequenced
was excised from the SDS PAGE gel and then proteolytically digested in situ in the
polyacrylamide gel slice.
[0049] Preparations of largely pure 36 kDa protein, that still had some minor contaminating
proteins, were loaded onto a 12% polyacrylamide gel. Three lanes each with 2 µg of
protein were loaded and electrophoresed in the gel until the dye front reached the
bottom of the gel. The gel was then stained in 0.2% uocmassie brilliant blue (w/v),
30% methanol (v/v), 1% acetic acid (v/v) for 20 minutes and then destain with 30%
methanol until the protein bands could be visualised. The 36 kDa band was identified
by comparison with molecular weight markers loaded into adjacent lanes and the band
from each lane was excised with a scalpel blade, taking care to exclude contaminating
bands.
[0050] The gel slices were transferred to a clean eppendorf tube and washed twice with 0.5ml
of 50% acetonitrile (v/v), 100mM Tris/Cl, pH 8.5. The washing removed some of the
uocmassie stain and also partially dehydrated the gel slices. The gel slices were
then removed from the tube and subjected to air drying on the laboratory bench until
they had shrunk significantly and started to curl up. They were then transferred back
to the eppendorf and rehydrated with firstly, 10µl of 100mM Tris/Cl, pH 8.5 containing
1µg of endoproteinase Lys C (Boehringer Mannheim). This is a proteinase that specifically
cleaves polypeptide chains on the carboxy terminal side of lysine residues. Further
Tris buffer was added to the gel slices until they were fully rehydrated and they
were then incubated at 37°C for 16 hours.
[0051] After incubation 1µl of trifluoroacetic acid was added to the tube to stop the reaction
and then the gel slices were washed twice with 0.3ml of 60% acetonitrile (v/v), 0.1%
TFA (v/v) at 30°C for 30 minutes. This was to again partially dehydrate the gel slices
causing them to shrink and elute the peptides that had been generated. The supernatant
was transferred to another clean eppendorf tube and then dried in a centrifugal evaporator
for 2 hours until the sample was near dryness and resuspended to a volume of 0.1ml
with 0.1% TEA.
[0052] The peptides were then separated by reversed phase HPLC on a Smart micropurification
system (Pharmacia). The peptide digest was loaded onto a C18 column (2.1 x 100 mm)
equilibrated in 0.1% TFA (Solvent A) at a flow rate of 0.1ml min. The column was theri
eluted with a gradient of 0 - 70% of Solvent B (90% acetonitrile v/v, 0.085% TFA v/v)
over 70 minutes at the same flow rate. The optical density was monitored at 214 nm
and individual peptide peaks were collected in the fraction collector by manual stepping.
Polypeptides were sequenced by loading onto a model 492 Perkin Elmer protein sequencer
using the liquid phase chemistry cycles as recommended by the manufacturer.
Example IV-a carrot cell culture
[0054] A carrot cell suspension culture line (NOR) was obtained from the Department of Biochemistry
and Molecular Biology, University of Leeds. The culture was maintained by subculturing
10 ml of the culture into 90 ml of fresh Murashige and Skoog medium (Sigma) containing
25 g/l sucrose and 1 mg/l 2,4-D every seven days. Cultures were incubated in an orbital
shaking incubator at 150 rpm at 25°C in the dark.
The NOR culture was cold treated as follows:
[0055] NOR cultures were transferred to 4°C after 4d and 7d of growth at 25°C. Cultures
were harvested at t=0, t=7d and t=14d. In addition to harvesting, the packed cell
volume (PCV) was determined for each culture at each time point.
[0056] The media samples from NOR suspension cultures were analyzed as follows. Approximately
1/10th of the volume of a frozen aliquot of conditioned suspension culture medium
was allowed to defrost. The defrosted (freeze concentrated) portion was removed and
tested for activity by sandwich splat assays as described in Example I. Medium from
cold acclimated cultures was found to contain significantly more activity than medium
from non-cold acclimated cultures.
[0057] The cold acclimated NOR carrot medium was buffered by addition of 100µl of 1M Tris/HCl
pH 7.4. Purification of activity was then performed by ion exchange and gel permeation
chromatography using a method based on that in Example II: the buffered medium was
applied to a 1 ml Q Sepharose column (Pharmacia) at a flow rate of 1 ml/min and bound
molecules eluted with 3 ml aliquots of 500 mM Tris/HCl pH 7.4 containing concentrations
of NaCl starting at 0.1 M and increasing to 0.5 M in 0.1 M steps. 1 ml fractions were
collected and tested for activity as in Example I.
[0058] The antifreeze activity in the active ion exchange fractions was purified by gel
permeation chromatography as follows. The active fraction from above was acetone precipitated
and the pellet resuspended in 50µl 50mM Tris/RCI + 0.15 M NaCl, pH 7.2. This was then
centrifuged at 10.000 g for 10 minutes, and 20µl loaded onto a Superdex 75 gel permeation
column on the Pharmacia SMART system. The flow rate was 40µl/min and the mobile phase
was 50mM Tris/HCl + 0.15M NaCl, pH 7.2. 80µl fractions were collected and splatted.
Activity was detected in fractions corresponding to a retention of 1.16 ml.
[0059] Further isolation of the active proteins can be done by SDS PAGE analysis in line
with Example II.
Example IV-b carrot root culture
[0060] Carrot root cultures were initiated as follows.
[0061] For each individual culture 10 surface sterilised
Daucus carote cv Autumn King seeds were placed into 100 ml MS medium containing 25 g/L sucrose
and 0.5 g/L MES in sterile 250 ml Erlenmyer flasks. Seeds were germinated by shaking
at 150 rpm in the dark at 25°C for 3 weeks. Leaves and shoots were then aseptically
removed. The roots were replaced into 100 ml fresh medium and incubated with shaking
for a further 2 weeks.
[0062] Homogenates were prepared from cold treated and non-cold treated root cultures as
follows. Fast frozen roots were ground up 3x in liquid nitrogen in a cold mortar and
pestle then transferred to a further chilled mortar and pestle and ground up with
0.5x volume of ice-cold 50mM Tris.HCl + 10 mM EDTA pH 7.4 containing 30 % w/w sucrose.
Homogenates were centrifuged at 10.000 g for 10 minutes at 4°C and the supernatant
tested for activity as in Example I. Significantly more activity was detected in cold
treated root cultures than in non-cold treated root cultures.
Example V preparation of ice-cream
[0063] Root extract from cold acclimatised carrot roots was prepared by scrubbing freshly
pulled cold acclimatised (as in example I) carrots in cold water. The tops are removed
and the juice extracted employing a domestic juice extractor (Russell Hobbs, model
no 9915). The juice was frozen in 1 litre blocks and stored a -20°C prior to collection
for use in ice cream trials. The carrot AFP juice was added to the following ice cream
formulation:
INGREDIENT |
parts by weight |
Skimmed Milk Powder |
10.000 |
Sucrose |
13.000 |
MD40 |
4.000 |
Locust Bean Gum |
0.144 |
Genulacta L100 |
0.016 |
MGP |
0.300 |
Butteroil |
8.000 |
Vanillin |
0.012 |
Water |
64.528 |
Carrot Extract (from cold acclimated carrots containing 1-10 mg AFP per kg) |
4.472 |
[0064] Ice-cream was prepared by freezing the above formulation and aeration to 106% overrun.
[0065] Measurements were made on fresh sample and on samples which had been abused by storage
at -10 °C for a period of 10 days. As a comparison a sample without carrot extract
was measured in the same way. The measurements were done as follows:
[0066] Samples were equilibrated at -18 °C in a Prolan Environmental cabinet for approximately
12 hours. Three samples were chosen representatively from each batch of ice cream
and a slide was prepared from each in a Cryostat temperature control cabinet by smearing
a thin layer of ice cream from the centre of each block onto a microscopic slide.
A single drop of white spirit was applied to the slide and a cover slip was then applied.
Each slide, in turn, was then transferred to a temperature controlled microscope stage
(Leit LaborLux S, Leica x10 objective, temperature -18 °C). Images of ice-crystals
(about 400 individual ice-crystals) were collected and relayed through a video camera
(Sanyo CCD) to an image storage and analysis system (LEICA Q520MC).
[0067] The stored ice crystal images were highlighted manually by drawing around the perimeter
which then highlights the whole crystal. Images of the highlighted crystals were then
measured using the image analysis software which counts the number of pixels required
to complete the longest straight line (length), shortest straight line (breadth),
the aspect ratio (length/breadth). The data for each individual ice crystal of a batch
of ice cream was imported into a spreadsheet where analysis of the data set was carried
out to find the mean, and standard deviation.
[0068] The ice Cream Hardness Measurements were carried out using a Hounsfield H10KM Universal
Tester, a Hounsfield 100N Load Cell and a 10cm Cylindrical Stainless steel probe.
The ice-cream samples were prepared by 16 Hour incubation of 486ml ice cream blocks
in a Prolan Temperature Control Cabinet set at -18 °C.
[0069] The ice cream block was removed from Prolan temperature control cabinet and placed
the Hounsfield H10KM Universal Tester. The 10cm cylindrical probe was pushed into
the ice cream block at a constant rate of 400mm/min to a depth of 20mm. The maximum
force recorded during the compression was used and expressed as the ice cream Hardness.
If cracking or brittle fracture of the sample was observed this was indicated in the
right hand column
[0070] The following results were obtained
|
Ice Crystal Size Parameters |
Material Properties |
Sample |
Mean Crystal Length / um |
Mean Crystal Breadth / um |
Mean Crystal Shape Factor / - |
Mean Crystal Aspect Ratio / - |
Hardness / N |
Brittle Fracture observation |
Carrot fresh |
26.79 ± AFP - 1.3 |
19.00 ± 0.9 |
1.15 ± 0.013 |
1.43 ± 0.024 |
40.8 |
Yes |
Carrot AFP - Abused |
33.48 ± 1.3 |
24.61 ± 0.9 |
1.13 ± 0.013 |
1.37 ± 0.020 |
59.9 |
Yes |
Cont.- Fresh |
33.67 ± 1.1 |
24.79 ± 0.8 |
1.12 ± 0.008 |
1.38 ± 0.018 |
27.3 |
No |
Cont.- Abused |
61.77 ± 2.7 |
46.54 ± 2.0 |
1.11 ± 0.010 |
1.37 ± 0.020 |
32.7 |
No |
[0071] This proves that carrot AFP has good ice recrystallisation inhibition properties.
Example VI
[0072] The peptide sequences shown in Example III were analyzed as to their suitability
for degenerate oligonucleotide primer design. Part of Peptide D (GLY-PRO-VAL-PRO-LEU-PHE-PHE-PRO)
was chosen and the primer cp3 (GGI CCI GTI CCI YTI TTY TTY CC, where I= inosine and
Y=C or T) was synthesized (Genosys) .
[0073] First strand cDNA was prepared from 5 µg cold acclimated (1 month as in example I)
carrot root RNA using Superscript Reverse Transcriptase (Stratagene) and an oligonucleotide
primer OG1 (GAGAGAGGATCCTCGAG(T)
15) according to the manufacturer's instructions. 1% of the first strand cDNA reaction
was used as template, together with cp3 and OG1 primers, in subsequent PCR. The reactions
were carried out in a thermal cycler using Taq DNA polymerase (Gibco BRL) for 30 cycles
(1 minute at 94 °C, 1 minute at 50 °C and 1 minute at 72 °C) according to the manufacturer's
instructions. All primers were used at a concentration of 1 µM. The resulting -800
bp PCR product was gel purified and cloned into the pTAg vector (R&D Systems) according
to the manufacturer's instructions. The cloned cp3 PCR product was sequenced using
the dideoxy sequencing method employed by the Sequenase kit (USB). The cp3 nucleotide
sequence and deduced amino acid sequence were substantially similar to:
[0074] In order to obtain the full coding region for the carrot AFP, a cDNA library was
constructed. A poly (A)+ quick column (Stratagene) was used to purify mRNA from 500
µg CA (1 month) carrot total RNA, according to manufacturer's instructions. All resulting
poly (A)+ RNA was used for cDNA synthesis and subsequent library construction using
the lambda ZAP vector kit (Stratagene). 1 x 10
5 recombinant phage clones were screened by hybridization using the cp3 PCR product
as a
32P labelled probe.
[0075] Positive plaques were screened to purity and phage-mids excised before the inserts
were characterised by DNA sequence analysis. Two cDNA clones were sequenced to completion.
Although the 5' and 3' untranslated regions contained some sequence variability, the
coding regions were identical. The coding regions of the two cDNA clones were substantially
similar to:
[0076] Partial sequence analysis of 4 other clones also indicated that they contained the
same coding region as the fully sequenced clones and thus all the positives from the
library screen were likely to represent transcripts from the same gene. The existence
of only one copy of the AFP gene in the carrot genome was further substantiated by
the fact that Southern analysis of restriction enzyme digested carrot genomic DNA
suggested that only one fragment hybridized to the probe.
Example VII
[0077] In order to prove that the carrot cDNA as shown in example VI represented an AFP,
expression of the coding region was carried out as follows. One of the cDNAs was first
cloned into an intermediate pUC plasmid vector (Messing, 1983) containing a double
CaMV
35S promoter (Guerineau, J. F., Woolsten, S., Brooks, L., and Mollineaux, P. (1988))
expression cassette, and then into a binary vector, as described below. All enzymes
used were supplied by Gibco BRL and used according to the manufacturer's instructions.
[0078] The pBluescript phagemid (Stratagene) containing the cDNA clone was digested with
Xho I and the recessed 3' termini filled in using the Klenow fragment of DNA polymerase
I. The cDNA fragment was then released from the vector by digestion with
Eco RI. The
Eco RI/blunt cDNA fragment was then cloned into the
Eco RI/blunt digested intermediate pUC plasmid vector. The CaMV
35S-cDNA expression cassette was then subcloned as a partial Hind III fragment into
Hind III-cut pBin 19 binary vector (Bevan 1984). The binary vector construct was then
introduced into tobacco using Agrobacterium mediated transformation (as described
in Draper, J., Scott, R., Armatage, P., and Walden, R. (1988)).
[0079] Transgenic tobacco callus was analyzed for expression of recrystallisation inhibition
activity as soon as sufficient kanamycin resistant material was regenerated. Small
scale protein extracts were made from several independent kanamycin resistant calli
plus some wild type tobacco callus. Approximately 2 g tissue was ground up in 1-2
mls sucrose buffer (30 % sucrose 50 mM Tris, 10 mM EDTA, 20 mM ascorbate, pH 7.2)
using a mortar and pestle. The solution was centrifuged at 10,000 xg for 2 minutes
and the supernatant removed to a fresh tube. An aliquot of 3 µl of protein extract
was tested for recrystallisation activity using the sucrose sandwich splat assay method
of example I. All kanamycin resistant callus extracts tested demonstrated recrystallisation
inhibition activity.
[0080] Stable transgenic tobacco plants expressing the carrot AFP have also been produced.
Leaf extracts from wild-type and transgenic tobacco plants have been subjected to
northern analysis using the AFP cDNA as a probe. The AFP message was only detectable
in the transgenic tobacco plants. This suggests that the AFP message is stable in
the greenhouse grown transgenic tobacco plants. When compared with the native carrot
transcript, the tobacco AFP transcript appears to be slightly bigger. This discrepancy
can be explained by the method of construction of the AFP expression cassette. Because
the
CaMV 35S polyadenylation signal is most 3' in the construct, it is likely that this signal
is used in the transgenic AFP message, giving rise to a longer transcript. Leaf extracts
from wild-type and transgenic tobacco plants have also been analyzed by western blotting
using a carrot AFP antibody. A cross-reacting protein was only detected in the transgenic
tobacco plants. Despite the difference in transcript size, the protein produced in
tobacco appears to be the same size as the native carrot AFP.
[0081] The above data provides the proof that the protein purified from carrot and the corresponding
cDNA represent an active AFP.
SEQUENCE LISTING
[0082]
- (1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: Unilever N.V.
(B) STREET: Weena 455
(C) CITY: Rotterdam
(E) COUNTRY: Netherlands
(F) POSTAL CODE (ZIP): 3013 AL
(G) TELEPHONE: 010-4605351
(H) TELEFAX: 010-4606290
(A) NAME: Unilever PLC
(B) STREET: Unilever House, Blackfriars
(C) CITY: London
(E) COUNTRY: United Kingdom
(F) POSTAL CODE (ZIP): EC4P 4BQ
(ii) TITLE OF INVENTION: Proteins
(iii) NUMBER OF SEQUENCES: 12
(iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.30 (EPO)
(v) CURRENT APPLICATION DATA:
APPLICATION NUMBER: PCT/EP97/06181
- (2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Daucus carota
(B) STRAIN: Autumn King
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
- (2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Daucus carota
(B) STRAIN: Autumn King
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
- (2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Daucus carota
(B) STRAIN: Autumn King
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
- (2) INFORMATION FOR SEQ ID NO: 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(v) FRAGMENT TYPE: internal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Daucus carota
(B) STRAIN: Autumn King
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
- (2) INFORMATION FOR SEQ ID NO: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Daucus carota
(B) STRAIN: Autumn King
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:
- (2) INFORMATION FOR SEQ ID NO: 6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 999 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Daucus carota
(B) STRAIN: Autumn King
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION:1..996
(C) IDENTIFICATION METHOD: experimental
(D) OTHER INFORMATION:/codon_start= 1
/product= "anti freeze protein"
/evidence= EXPERIMENTAL
/number= 1
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
- (2) INFORMATION FOR SEQ ID NO : 7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 332 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:
- (2) INFORMATION FOR SEQ ID NO: 8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Daucus carota
(B) STRAIN: Autumn King
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:
- (2) INFORMATION FOR SEQ ID NO: 9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "primer cp3"
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 1..12
(D) OTHER INFORMATION:/note= "N is used to indicate inosine(i)"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:
- (2) INFORMATION FOR SEQ ID NO: 10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "primer OG1"
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:
- (2) INFORMATION FOR SEQ ID NO: 11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 829 base pairs
(B) TYPE: nucleic acid
(C) SIRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "cp3 nucleotide sequence (see patent application page 26/27)"
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Daucus carota
(B) STRAIN: Autumn King
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION:1..591
(D) OTHER INFORMATION:/codon_start= 1 /product= "anti freeze protein" /number= 1
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:
- (2) INFORMATION FOR SEQ ID NO: 12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 197 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:
1. A polypeptide having antifreeze activity which has an amino acid sequence as represented
in Listing I, and isoforms or derivatives thereof which show at least 75% homology
with the amino acid sequence of Listing I and which still possess antifreeze activity.
2. A polypeptide having antifreeze activity which has an amino acid sequence as represented
in Listing I, and isoforms or derivatives thereof which show more than 85% homology
with the amino acid sequence of Listing I and which still possess antifreeze activity.
3. A polypeptide having antifreeze activity which has an amino acid sequence as represented
in Listing I.
4. An isolated nucleic acid sequence encoding the polypeptide of any one of claims 1
to 3.
5. An isolated nucleic acid sequence comprising the nucleotide sequence as represented
in Listing I.
6. A method of obtaining a polypeptide according to any one of claims 1 to 3, whereby
the polypeptide is isolated from cold-acclimatised carrots.
7. A method of obtaining a polypeptide according to any one of claims 1 to 3, whereby
the polypeptide is expressed by a genetically modified non-human organism.
8. A method according to claim 7, whereby the organism is a microorganism, a plant or
a cell culture.
9. An antibody capable of specifically binding the polypeptide of any one of claims 1
to 3.
10. A food product comprising a polypeptide of any one of claims 1 to 3 with the proviso
that the food product is not a carrot.
11. The food product of claim 10 being a frozen confectionery product or a frozen vegetable.
12. A method of producing a food product comprising an antifreeze polypeptide according
to any one of claims 1 to 3 comprising the steps of
(a) adding to the food product a composition comprising said antifreeze polypeptide;
or
(b) in situ production of said antifreeze polypeptide.
13. Use of the polypeptide of any one of claims 1 to 3 for increasing the frost tolerance
of plants.
14. A microorganism, cell line or plant transformed by a gene construct that encodes the
polypeptide of any one of claims 1 to 3 with the proviso that the plant is not an
unmodified carrot plant.
1. Polypeptide ayant une activité antigel qui possède une séquence d'acides aminés telle
que représentée dans le listage 1, et des isoformes ou des dérivés de ceux-ci qui
montrent au moins une homologie de 75% avec la séquence d'acides aminés du listage
1 et qui possèdent encore une activité antigel.
2. Polypeptide ayant une activité antigel qui possède une séquence d'acides aminés telle
que représentée dans le listage 1, et des isoformes ou des dérivés de ceux-ci qui
montrent une homologie de plus de 85% avec la séquence d'acides aminés du listage
1 et qui possèdent encore une activité antigel.
3. Polypeptide ayant une activité antigel qui a une séquence d'acides aminés telle que
représentée dans le listage 1.
4. Séquence d'acides nucléiques isolée codant le polypeptide de l'une quelconque des
revendications 1 à 3.
5. Séquence d'acides nucléiques isolée comprenant la séquence de nucléotides telle que
représentée dans le listage 1.
6. Procédé d'obtention d'un polypeptide selon l'une quelconque des revendications 1 à
3, de manière à ce que le polypeptide soit isolé à partir de carottes acclimatées
au froid.
7. Procédé d'obtention d'un polypeptide selon l'une quelconque des revendications 1 à
3, de manière à ce que le polypeptide soit exprimé par un organisme non humain génétiquement
modifié.
8. Procédé selon la revendication 7, moyennant quoi l'organisme soit un microorganisme,
une plante ou une culture cellulaire.
9. Anticorps capable de se lier spécifiquement au polypeptide selon l'une quelconque
des revendications 1 à 3.
10. Produit alimentaire comprenant un polypeptide selon l'une quelconque des revendications
1 à 3, sous réserve que le produit alimentaire ne soit pas une carotte.
11. Produit alimentaire selon la revendication 10, qui est un produit de confiseries congelé
ou un légume congelé.
12. Procédé de production d'un produit alimentaire comprenant un polypeptide antigel selon
l'une quelconque des revendications 1 à 3 comprenant les étapes consistant à
(a) ajouter au produit alimentaire une composition comprenant ledit polypeptide antigel
; ou
(b) produire in situ ledit polypeptide antigel.
13. Utilisation du polypeptide selon l'une quelconque des revendications 1 à 3, afin d'augmenter
la tolérance au gel des plantes.
14. Microorganisme, lignée cellulaire ou plante transformé par un gène chimère qui code
le polypeptide selon l'une quelconque des revendications 1 à 3, sous réserve que la
plante ne soit pas un plant de carottes non modifiées.
1. Polypeptid mit Frostschutz-Aktivität, welches eine Aminosäure-Sequenz, wie in Aufzählung
I dargestellt, hat, und Isoformen oder Derivate davon, die mindestens 75% Homologie
mit der Aminosäure-Sequenz der Aufzählung I zeigen und die noch immer Frostschutz-Aktivität
besitzen.
2. Polypeptid mit Frostschutz-Aktivität, welches eine Aminosäure-Sequenz, wie in Aufzählung
I dargestellt, hat, und Isoformen oder Derivate davon, die mehr als 95% Homologie
mit der Aminosäure-Sequenz der Aufzählung I zeigen und die noch immer Frostschutz-Aktivität
besitzen.
3. Polypeptid mit Frostschutz-Aktivität, welches eine Aminosäure-Sequenz, wie in Aufzählung
I dargestellt, hat.
4. Isolierte Nukleinsäure-Sequenz, die für das Polypeptid nach einem der Ansprüche 1
bis 3 codiert.
5. Isolierte Nukleinsäure-Sequenz, umfassend die in Aufzählung I dargestellte Nukleotid-Sequenz.
6. Verfahren zum Erhalt eines Polypeptids nach einem der Ansprüche 1 bis 3, wobei das
Polypeptid aus an Kälte akklimatisierten Karotten isoliert wird.
7. Verfahren zum Erhalt eines Polypeptids nach einem der Ansprüche 1 bis 3, wobei das
Polypeptid durch einen genetisch modifizierten, nicht-humanen Organismus exprimiert
wird.
8. Verfahren nach Anspruch 7, wobei der Organismus ein Mikroorganismus, eine Pflanze
oder eine Zellkultur ist.
9. Antikörper, welcher das Polypeptid eines der Ansprüche 1 bis 3 spezifisch binden kann.
10. Nahrungsmittelprodukt, umfassend ein Polypeptid eines der Ansprüche 1 bis 3, mit der
Maßgabe, dass das Nahrungsmittelprodukt keine Karotte ist.
11. Nahrungsmittelprodukt nach Anspruch 10, welches ein gefrorenes Konfekt-Produkt oder
ein gefrorenes Gemüse ist.
12. Verfahren zur Herstellung eines Nahrungsmittelprodukts, umfassend ein Frostschutz-Polypeptid
nach einem der Ansprüche 1 bis 3, welches die Schritte umfasst:
(a) Zugeben einer das Frostschutz-Polypeptid umfassenden Zusammensetzung zum Nahrungsmittelprodukt,
(b) in situ-Produktion des Frostschutz-Polypeptids.
13. Verwendung des Polypeptids eines der Ansprüche 1 bis 3 zur Erhöhung der Gefrier-Toleranz
von Pflanzen.
14. Mikroorganismus, Zelllinie oder Pflanze, transformiert durch ein Gen-Konstrukt, das
für das Polypeptid eines der Ansprüche 1 bis 3 codiert, mit der Maßgabe, dass die
Pflanze keine unmodifizierte Karotten-Pflanze ist.